1 INTRODUCTION
Porcine epidemic diarrhea virus (PEDV), a member of the genusAlphacoronavirus in the family Coronaviridae of the orderNidovirales , is a main etiology causing porcine epidemic diarrhea (PED) which is a devastating disease characterized by acute watery diarrhea, vomiting, dehydration, and high mortality in neonatal piglets. PEDV was first reported in England and Belgium (Wood, 1977, Pensaert and de Bouck, 1978) in the late 1970s. Outbreaks of PED in China were sporadic (Chen et al., 2010) until late 2010, when PEDV variant strains-caused PED broke out and spread throughout the country (Wang et al., 2013; Sun et al., 2012). PEDV has also been reported in the US (Stevenson et al., 2013) and other countries in North America, including Canada and Mexico (Mole, 2013, Ojkic et al., 2015, Trujillo-Ortega et al., 2016). PEDV has caused considerable economic damage to the global pig industry.
The PEDV genome is a single-stranded positive –sense RNA with approximately 28kb in length, and encodes four structural proteins which include spike (S), nucleocapsid (N), envelop (E), and membrane (M) protein. Among these proteins, the S protein is on the surface of the PEDV virion that plays a pivotal role in regulating interactions with specific host cell receptors to mediate viral entry, inducing neutralizing antibodies in the natural host and determining antigenicity and virulence (Li et al., 2020, Suzuki et al., 2018, Lv et al., 2016, Cruz et al, 2008, Sun et al.,2008, Park et al., 2007) . The S protein is often used to evaluate the genetic diversity of PEDV strains (Li et al., 2012; Lee et al.,2014). Based on the phylogenetic analysis of the S gene, the current reported PEDV strains could be genetically divided into two genotypes: G1 and G2. PEDV G1 and G2 can be further divided into G1a, G1b, and G2a, G2b subtypes, respectively. The G1 contains classical strains, including the prototype strain CV777 and historical vaccine strains. The G2 contains the post-2010 global epidemic isolates (Jung at al., 2020, Won et al., 2020).
The fact of increased outbreaks of G2 PEDV and the failure of G1 PEDV strain (CV777)-based vaccine in China since 2010 implied that the difference of antigenicity between G1 and G2 PEDV strains. Identification of factors contributing to the antigenicity difference between G1 and G2 PEDV strains will lead to the development of an effective PEDV vaccine with better protection against prevalent genotype of PEDV. As amino acid mutations that distinguish the two PEDV genotypes were mostly located in the N-terminal domain (NTD) (aa 1-380) of S protein, the objective of this study is to determine the role of the NTD of S protein in resulting antigenicity difference between PEDV G1 and G2. The NTD of the S protein from CV777 vaccine strain (G1) and CH/ZMDZY/11 strain (G2) was expressed in E. coli , respectively. Polyclonal antibodies (PAbs) against genotype-specific S proteins were prepared by immunizing BALB/c mice using purified S proteins. Antigenicity was systematically compared by detection of PAbs against two genotype PEDV strains and purified S proteins using Western blot, indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and serum cross-neutralization assay (SN).